1. Field of the Invention
The invention relates to methods for culturing human embryonic stem (HES) cells. More particularly, to methods for culturing single HES cells in a substantially undifferentiated state.
2. Description of Related Art
Human embryonic stem (HES) cells are pluripotent cell lines that have been derived from the inner cell mass (ICM) of blastocyst stage embryos. HES cells have the potential to develop into any type of cells and to generate any types of tissues, organs or body parts, including a whole organism. As such, it is expected that the ability to provide normal clonal HES cells on demand and to manipulate the differentiation thereof will provide a powerful tool capable of driving radical advances in the biomedical, industrial and scientific fields.
Potential applications of HES cells are far ranging and include drug discovery and testing, generation of cells, tissues and organs for use in transplantation, production of biomolecules, testing the toxicity and/or teratogenicity of compounds and facilitating the study of developmental and other biological processes. For example, diseases presently expected to be treatable by therapeutic transplantation of HES cells or HES-derived cells include Parkinson's disease, cardiac infarcts, juvenile-onset diabetes mellitus, and leukemia (Gearhart J. Science 282: 1061-1062, 1998; Rossant and Nagy, Nature Biotech. 17: 23-24, 1999).
There are, however, significant hurdles to the practical exploitation of HES cells.
To maintain HES cells in an undifferentiated state, HES cells are usually cultured on feeder cells. The feeder cells can secrete factors needed for stem cell self-renewal and proliferation, while at the same time, inhibit their differentiation.
Commonly used feeder cells includes a primary mouse embryonic fibroblast (PMEF), a mouse embryonic fibroblast (MEF), a murine fetal fibroblast (MFF), a human embryonic fibroblast (HEF), a human fetal muscle cell (HFM), a human fetal skin cell (HFS), a human adult skin cell, a human foreskin fibroblast (HFF), a human adult fallopian tubal epithelial cell (HAFT) and a human marrow stromal cells (hMSCs) (International Patent Publication Nos. WO 03/02944 and 03/014313; J. H. Park et al., Biol Reprod. 69:2007-2017, 2003; M. Amit et al., Biol Reprod. 68 (6):2150-2156, 2003; Outi Hovattal et al., Hum. Reprod. 18 (7): 1404-1409, 2003; Richards, M. et. al, Nat Biotechnol. 20(9):933-936, 2002; James A. et al., Science 282 (6):1145-1147, 1998; Linzhao Cheng et al., Stem Cells 21:131-142, 2003).
The HES cells can also be cultured on an extracellular matrix (ECM) instead of feeder cells.
Commonly used matrix includes the basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma (e.g. Matrigel™), or bovine-fibronectin/laminin. These matrix are usually supplemented with a mouse embryonic fibroblast (MEF) conditioned medium, or a synthetic medium supplemented with bovine serum and growth factors (Xu et al. Nat. Biotechnol. 19 (10):971-974, 2001; International Patent Publication No. WO 03/020920; U.S. patent Pub. No. 20030017589).
Once established and expanded, HES cells are routinely passaged by manual dissociation. Unlike mouse embryonic stem cells, the HES cells cannot be enzymatically dispersed into single cells without causing undesirable death or differentiation. When the sizes of colonies reach 1-to-2 mm diameter, they are cut into several pieces with a pulled glass pipette and the colony pieces are inoculated onto new feeder cells or extracellular matrix (ECM) for sub-culturing. Colony piece sizes of about 50 to 100 cells were optimal.
However, such culture method for HES cells described above introduces disadvantages in the scaling up and downstream manipulation and experimentation of HES cells. Some of these disadvantages are (1) the labor intensiveness in having to passage by manual dissociation, unlike other cells that can be passaged by exposure to enzymes; (2) the constraints of scaling up large numbers of HES cells because the propagation of undifferentiated HES cells can not be achieved by utilizing single cells; (3) a small cluster of HES cells makes the transfection procedure more difficult than that of single cells and therefore complicates both experimental procedures and results.
To establish culture conditions that allow the growth and survival of single undifferentiated HES cells is therefore an urgent necessity to help overcome these disadvantages and lead the prospects of large/bulk scale HES cell production.
An article by Amit et al. (Dev. Biol. 227: 271-278,2000; U.S. patent Pub. No. 20030073234) is entitled Clonally Derived Human Embryonic Stem Cell Lines Maintain pluripotency and Proliferative Potential for Prolonged Periods of Culture Although this publication reported that HES cell line H9 could be subcloned as single cells on feeder cells, our data indicate that HES-3 lines could not be propagated by using such method.
There is need, therefore, to provide an alternative method for culturing single HES cells.